<meta charset="utf8"></head><body>뇌졸중 후 이소성 neurod1 발현을 연구하기 위한 AAV 시스템 및 마우스 모델

<ol>
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	<tbody>
		<tr >
			<td >#77 Drill Bit (.018&#8221;)</td>
			<td >David Kopf Instruments</td>
			<td >8177</td>
			<td ></td>
		</tr>
		<tr >
			<td >AAV5-GFAP(0.7)-mNeurod1-2A-iCre</td>
			<td >Vector&#160;Biolabs</td>
			<td ></td>
			<td></td>
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		<tr >
			<td >Absorbable Suture with Needle Polysorb&#8482; Polyester CV-15 3/8 Circle Taper Point Needle Size 4 - 0 Braided</td>
			<td >Covidien</td>
			<td >GL-881</td>
			<td></td>
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			<td >Anti-NeuN Antibody (rabbit)&#160;</td>
			<td >Millipore Sigma</td>
			<td >ABN78&#160;</td>
			<td></td>
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		<tr >
			<td >C57BL/6 Mice</td>
			<td >Charles River</td>
			<td >027</td>
			<td></td>
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			<td >Chlorhexidine Solution</td>
			<td >Partnar</td>
			<td >PCH-020</td>
			<td></td>
		</tr>
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			<td >Contec&#8482;&#160;PREempt&#8482; RTU Disinfectant Solution</td>
			<td >Fisher Scientific</td>
			<td >29-636-6212</td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat</td>
			<td >Thermo Scientific&#160;</td>
			<td >HM525 NX&#160;</td>
			<td></td>
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			<td >Endothelin 1</td>
			<td >Millipore Sigma</td>
			<td >05-23-3800-0.5MG</td>
			<td></td>
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		<tr >
			<td >Feather Safety Razor Microtome Blades</td>
			<td >Feather</td>
			<td >12-631P</td>
			<td></td>
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			<td >Fisherbrand&#160;Cover Glasses: Rectangles</td>
			<td >Fisher Scientific</td>
			<td >12-545M&#160;</td>
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			<td >Fluorescence Mounting Medium</td>
			<td >Agilent Technologies</td>
			<td >S3023&#160;</td>
			<td></td>
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			<td >Hamilton Syringe</td>
			<td >Hamilton Company</td>
			<td >7634-01</td>
			<td></td>
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			<td >High Speed Stereotaxic Drill</td>
			<td >David Kopf Instruments</td>
			<td >1474</td>
			<td></td>
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			<td >Metacam Solution (Meloxicam)</td>
			<td >Boehringer Ingelheim</td>
			<td ></td>
			<td></td>
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		<tr >
			<td >O.C.T Compound</td>
			<td >Fisher Scientific</td>
			<td >23-730-571</td>
			<td></td>
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			<td >pAAV2/5-GFAP-iCre</td>
			<td >Vector Builder</td>
			<td >P190924-1001suq</td>
			<td></td>
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			<td >Polyderm Ointment USP</td>
			<td >TARO</td>
			<td >2181908</td>
			<td></td>
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		<tr >
			<td >SOMNI Scientific&#8482;&#160;The Animal Temperature Heating Pad</td>
			<td >Fisher Scientific</td>
			<td >04-777-177</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereotaxic Instruments</td>
			<td >David Kopf Instruments</td>
			<td >Model 902</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Plus Microscope Slides, white</td>
			<td >Fisherbrand</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >tdTomato Reporter Mice&#160;</td>
			<td >The Jackson Laboratory</td>
			<td >007914</td>
			<td></td>
		</tr>
		<tr >
			<td >V-1 Tabletop Laboratory Animal Anesthesia System</td>
			<td >VetEquip</td>
			<td >901806</td>
			<td></td>
		</tr>
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aav systems and mouse models for investigating ectopic expression of neurod1 in transduced cells at subacute and chronic times post-ischemic stroke

<meta charset="utf8"></head><body>허혈성 뇌졸중 후 아급성 및 만성 시기에 형질도입된 세포에서 Neurod1의 이소성 발현을 조사하기 위한 AAV 시스템 및 마우스 모델

허혈성 뇌졸중 후 아급성 및 만성 시기에 형질도입된 세포에서 Neurod1의 이소성 발현을 조사하기 위한 AAV 시스템 및 마우스 모델

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic ...

aav-systems-mouse-models-for-investigating-ectopic-expression-neurod1

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JoVE Journal

Neuroscience

AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

312 조회수.  University of Toronto. 이 프로토콜은 피질 허혈성 뇌졸중 후 Neurod1의 바이러스 매개 이소성 발현을 설명합니다. Neurod1은 (1) 뇌졸중 후 아급성기(7일) 동안 야생형 마우스에서 Cre-Flex AAV 시스템을 사용하여 전달되고 (2) 뇌졸중 후 만성기(21일) 동안 조건부 리포터 마우스에서 단일 AAV 벡터를 사용하여 전달됩니다.

비디오: 허혈성 뇌졸중 후 아급성 및 만성 시기에 형질도입된 세포에서 Neurod1의 이소성 발현을 조사하기 위한 AAV 시스템 및 마우스 모델

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

연구

신경과학

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

마우스 두피 Astrocytes의 분리와 문화

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

측면 만성 두개골 창 준비 활성화<em&gt; 생체</em&gt; 마우스의 말초 중간 대뇌 동맥 폐색에 따라 관측

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

대뇌 혈관 내피 세포의 생체 에너지 기능의 평가

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

Vivo에서 실크/AAV 영화를 사용 하 여 Optogenetic 단백질의 표적으로 표현

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

쥐에서 만성 이식을 위한 말초 신경 자극 전극의 준비

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors

Trunk compensation is the most common movement strategy to substitute for upper extremity (UE) motor deficits in chronic stroke survivors. There is a lack of evidence examining how task conditions impact trunk compensation and goal-directed arm reaching kinematics. This protocol aims to investigate the impact of task conditions, including task difficulty and complexity, on goal-directed arm reaching kinematics. Two non-disabled young adults and two chronic stroke survivors with mild UE motor impairment were recruited for testing the protocol. Each participant performed goal-directed arm reaches with four different task conditions (2 task difficulties [large vs. small targets] X 2 task complexities [pointing vs. picking up]). The task goal was to reach and point at a target or pick up an object located 20 cm in front of the home position as quickly as possible with a stylus or a pair of chopsticks, respectively, in response to an auditory cue. Participants performed ten reaches per task condition. A 3-dimensional motion capture camera system was used to record trunk and arm kinematics. Representative results showed that there was a significant increase in movement duration, movement jerkiness, and trunk compensation as a function of task complexity, but not task difficulty in all participants. Chronic stroke survivors showed significantly slower, jerkier, and more feedback-dependent arm reaches and significantly more compensatory trunk movements than non-disabled adults. Our representative results support that this protocol can be used to investigate the impact of task conditions on motor control strategies in chronic stroke survivors with mild UE motor impairment.

This protocol is intended to investigate the impact of task conditions on movement strategies in chronic stroke survivors. Further, this protocol can be used to examine if a restriction in elbow extension induced by neuromuscular electrical stimulation causes trunk compensation during goal-directed arm reaches in non-disabled adults.

만성 뇌졸중 생존자의 운동학 및 트렁크 보상에 도달하는 목표 지향 팔에 대한 모터 작업 조건의 영향

Trunk compensation is the most common movement strategy to substitute for upper extremity (UE) motor deficits in chronic stroke survivors. There is a lack ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

마우스에서 뇌졸중 모델링: 포심 피질 병변

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

마우스에서 뇌졸중 모델링: 외부 경동맥을 통한 일시적인 중간 뇌동맥 폐색

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

마우스 뇌 에서 생체 내 인핸서 기반 발현 구조체의 AAV 배포

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...